We are continuing our efforts to immunolocalize proteins in both mammalian cells and in yeast by applying antibodies to thin sections of samples that have been well-fixed by techniques of cryopreservation and embedded in Lowicryl or LR-White. Of 13 antibodies obtained from potential collaborators, only 4 have yielded well-controlled localizations with signals that are above background by statistically rigorous tests. We have had limited success using epitope tagged versions of the protein products of genes from the yeast Schizosaccharomyces pombe, but have failed to localize other types of epitope tags. We are now experimenting with different protocols for freeze-substitution where no chemical crosslinkers are used prior to low temperature embedding or substituting but with only the addition of uranyl acetate to enhance membrane profiles. These preparations are of reasonably high quality, and we are proceeding to test the reactivity of fixative-sensitive antibodies at the surface of sections cut from this material. Our ability to cut ribbons of thin (10-30nm) serial sections is allowing us to gather data at a larger surface-to-volume ratio, which is important for antigens that are present at low abundance or for antigens that localize to small structures.